The functional atlas of nuclear receptors (NRs) sets out to obtain novel insights into the regulation of the transcriptional activity of NRs in their unliganded and liganded states. Our laboratory has been especially focused on the mechanism of repression by unliganded NRs, using as models the thyroid hormone receptor, the retinoic acid receptor, and the orphan receptor RevErb. We have been extremely interested in protein interactions made by N-CoR and the related silencing mediator of retinoid and thyroid receptors (SMRT). Our work, and that of others, has suggested that numerous proteins interact with N-CoR and SMRT, including a variety of histone deacetylases (HDACs) and other potential transcriptional mediators including mSin3, SUN-CoR, ETO, TBL1. We hypothesize that critical NR repression complexes vary as a function of cell type and environment. The goal of the present project is to utilize a proteomic approach to characterize these potential NR repression complexes, and to utilizes genomic, bioinformatics, and ultimately proteomics to determine their target genes in various cell types under different conditions. Specific Aim 1 is to create and characterize stable cell lines over-expressing N-CoR and SMRT. We and others have found numerous other proteins with the potential to interact with N- CoR/SMRT. To determine the importance of these other interactions in different cell types, we will stably over-express epitope tagged-versions of SMRT and N-CoR in multiple cell types, including HeLa cells, human embryonic kidney cells, myoblasts, preadipocytes, hematopoietic, and teratoma cells. Complexes will be purified biochemically and evaluated proteomically. Specific Aim 2 is to create and characterize stable cell lines over-expressing co-repressing co-reppressor interacting proteins. N- CoR and SMRT interact with a variety of histone deacetylases (HDACs) and other potential transcriptional mediators that may have important functions in co-repressor complexes, and these proteins will be systematically epitope tagged and stably expressed in the same cell type as in Aim 1, and their biochemical complexes will be evaluated proteomically. Specific Aim 3 is to delineate co-repressor gene targets in living cells. Co-repressors do not directly interact with target genes, but are recruited by NRs. To begin to catalog and understand their range of target genes, chromatin immunoprecipitation (ChIP) of N-CoR and SMRT from wild type cells as well as the stable lines will be performed, and libraries of associated gene fragments will be created, sequenced, and annotated. Bioinformatic analysis of such data from multiple cell types will provide great insight into the commonalities and differences of gene targets for these NR co-repressors. Together, these studies will provide critical information about NR co-repressor complexes and target in genes in various cell types, that be of great interest to the field and comprise an important component of the functional atlas of nuclear receptors.